Over the past two decades, there has been increasing concern about the possible effects of exposure to chemicals in the environment on endocrine and reproductive systems in humans and wildlife (Kavlock et al. 1996). To address these concerns, national and international programs have been initiated to develop new guidelines for the screening and testing of potential endocrine-disrupting chemicals (EDCs) in vertebrates. The Safe Drinking Water Act Amendments of 1995 and the Food Quality Protection Act of 1996 mandate screening for endocrine-disrupting properties of chemicals in drinking water or pesticides used in food production. In response to this legislation, the federal Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) has identified disrupting the process of steroidogenesis as one of the important toxicity pathways of endocrine disruption in addition to binding to three key endocrine nuclear receptors, i.e. estrogen receptors (ER), androgen receptors (AR) and thyroid hormone receptor (ThR) (Hilscherova et al. 2004; Sanderson et al. 2002; Zhang et al. 2005). The human H295R adrenocarcinoma cell-based steroidogenesis assay has been approved by the United States Environmental Protection Agency (USEPA) for use in Tier I of the Endocrine Disruptor Screening Program (EDSP) and is currently in the last phase of validation through OECD as an international standard to test chemicals for endocrine disrupting effects. While previously used assays have used production of different mRNAs as endpoints, currently used assays use as endpoints the production and release to the medium of the steroid hormones testosterone (T) and 17β-estradiol (E2) (Hecker et al. 2006).
H295R cells express genes that encode for all the key enzymes involved in steroidogenesis (FIG. 1) (Gazdar et al. 1990; Staels et al. 1993; Rainey et al. 1994). This is a unique property, because in vivo expression of these genes is tissue- and developmental stage-specific with no one tissue or developmental stage simultaneously expressing all of the genes involved in steroidogenesis. H295R cells have physiological characteristics of zonally undifferentiated human fetal adrenal cells. H295R cells represent a unique in vitro system with the ability to produce the steroid hormones found in the adult adrenal cortex and the gonads, which allows testing for effects on both corticosteroid synthesis and the production of sex steroid hormones such as androgens and estrogens.
One of the key hormones of interest, E2, is produced by H295R cells at relatively small and varying concentrations (˜10-50 pg E2/ml in culture medium) that are difficult to measure by use of automated ELISA or the more laborious LC\MS-MS method. Concentrations of E2 released by H295R cells into the medium are near the current limit of quantification (LOQ, approximately 2-10 pg E2/ml), which makes it difficult to measure reductions in E2 release caused by EDCs. The relatively great variance in E2 production around the detection limit is also a limiting factor. In addition, due to the small basal concentrations released by the H295R cells into the medium, it is difficult to demonstrate a decrease in production, which is also important for use as a screening tool. This is especially true with regard to the assessment of weak inhibitors.
Another endpoint of interest in screening for potential endocrine disruption is changes in expression of the aromatase (CYP19) gene, protein and enzyme activity, the enzyme that transforms (aromatizes) Testosterone to E2.